In human infertility practice, the classical in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) technologies are still used after superovulation with expensive hormone therapies to obtain large follicles (i.e at least 17 mm diameter). Both the costs of the hormones, their associated risk for acute and long term complications and the inconvenience of repeated hospital visits to monitor follicle growth incite the development of a less costly and better tolerated therapeutic procedure for infertile couples with a normal/high follicular reserve.
Oocyte in vitro maturation (IVM) is a technique that enables to mature germinal vesicle (GV)—stage oocytes that are enclosed in a firmly condensed corona cumulus layer (cumulus oocyte complexes (COCs)). These oocytes can be obtained by ultrasound-guided needle puncture before ovulation has been triggered by administration of human chorionic gonadotrophin (hCG) or early after hCG administration. Therefore, oocyte IVM has the potential to simplify fertility treatment or to reduce risks and costs related to hormone (hCG) stimulation in patients with normal or high ovarian follicular reserve. This follicular reserve is routinely determined by measurement of the anti-müllerian hormone levels and by ultrasound-guided follicle count on day 3 of the menstrual cycle.
For oocyte IVM, using ultrasound-guided needle puncture, the oocytes can be collected from small (<10 mm) follicles, in minimally stimulated or unstimulated ovaries and matured in vitro. However, interfering with the normal development of the oocyte at an early stage of development (e.g. when the follicle has only attained a diameter of 10 mm or less) not only produces fewer matured oocytes, but also decreases subsequent embryo development and implantation. In particular, successful implantation rate per embryo following IVM is usually less than 10% and is only half as successful as the rate that is reported for a routine IVF or ICSI procedure. The rate of early pregnancy loss seems to be variable, but is generally greater than after IVF/ICSI. For human patients, the reduced meiotic maturation rate (50%) currently forms a major bottleneck of current IVM technologies, together with the observed deficiency in embryo development of the matured oocytes (De Vos et at, Fertil. Steril. 2011; 96(4) 860-864; Guzman et al, Fertil. Steril. 2012; 98(2): 503-507). Therefore, the reduced developmental potential and implantation rate of IVM embryos needs to be addressed before the method can become widely accepted.
The cornerstone of the IVM culture is the provision of an appropriate environment for the attainment of developmental competence. This requires mainly hormonal intervention and the activation of necessary signaling pathways by using chemical compounds that allow synchronization of nuclear and cytoplasmic maturation processes within the oocyte. The rationale of a prolonged oocyte maturation period in vitro is to promote a longer interaction between the immature oocyte with adequately conditioned cumulus cells.
The signaling pathways between follicular cells and oocytes responsible for meiotic arrest have been extensively investigated. Meiotic arrest is maintained by production of high levels of cyclic adenosine monophosphate (cAMP). Intra-oocyte cAMP concentration is regulated by the activity of phosphodiesterase (PDE) enzymes that degrade cAMP. Recent studies showed that cGMP is produced in the cumulus cells upon activation of the guanilyl-cyclase coupled natriuretic peptide receptor type-2 (NPR2). NPR2 activity is induced by its ligand natriuretic peptide precursor C (NPPC), which is mainly synthesized by mural granulosa cells and cleaved into the C-type natriuretic peptide (CNP). cGMP is then transferred to the oocyte where it Inhibits the hydrolysis of CAMP by the phosphodiesterase PDE3A. This inhibition maintains a high concentration of cAMP and thus blocks meiotic progression. (Tsafriri et at Dev. Biol. 1996, 178(2): 393-402; Conti et al, Mol. Cell. Endocrinol. 1998, 145(1-2): 9-14; Conti et al., Mol. Cell. Endocrinol. 2002, 187(1-2): 153-159).
Pharmacological interference with the cAMP levels in the in vitro matured oocyte, derived from different species such as mouse, bovine, human, has been previously attempted in order to promote oocyte meiotic arrest, and allow for the acquisition of oocyte competence (Nogueira et al. Biol. Reprod. 2003, 69(6): 2045-2052; Thomas et at Biol. Reprod. 2004, 71(4): 1142-1149; Shu et at Hum. Reprod. 2008, 23(3): 504-513; Vanhoutte et ai. Hum. Reprod. 2009, 24(3); 658-669). Nevertheless, no major improvements have been reported at the level of embryonic developmental potential.
Recently, the CNP/NPR2 signaling pathway has shown to be a crucial regulatory mechanism in the maintenance of oocyte meiotic arrest in mid-size and fully-grown follicles (Zhang et al. J. Cell, Physiol. 2015, 230(1): 71-81; Sato et al. Mol. Endocrinol. 2012, 26: 1158-1166, Franciosi et al. Biol. Reprod. 2014, 91(3): 61, Santiquet et al. Biol. Reprod. 2014, 91(1): 16). However, the major problem in the above-mentioned studies is that the COCs can only be kept for a short time in meiotic arrest, and hence, in vitro maturation of only mid-size to large follicles was successful and maturation of small early antral follicles failed. Therefore, it is currently a major challenge to delay the start of meiotic resumption of small early antral follicles as well in order to improve their IVM process. In particular, such a protocol should not affect the further in vitro maturation. This challenge is even higher for such small early antral follicles as they need to gain or retain the capability of completing the nuclear maturation (i.e. moving from non-surrounded nucleolus (NSN) into surrounded nucleolus (SN) stage) before moving into the next steps of potential development, or even keeping the interconnection between oocytes and cumulus, allowing for the transfer of the nutrients from cumulus (e.g. RNA cargo)).